Nucleic acid amplification, and the polymerase chain reaction (PCR) in particular, is an important research tool, with applications in cloning, analysis of genetic expression, DNA sequencing, genetic mapping, drug discovery, and the like (Gilliland etal, (1990) Proc. Natl. Acad. Sci., 87:2725-2729; Bevan etal, (1992) PCR Methods and Applications, 1:222-228; Green etal, (1991) PCR Methods and Applications, 1:77-90). Descriptions of, and guidance for conducting, PCR are provided in extensive literature (Innis etal, (1989) in PCR Protocols, Academic Press, NY; McPherson etal, (1991) in PCR: A Practical Approach, Volume 1, Oxford University Press, Oxford, pp. 46, 199; McPherson etal, (1995) in PCR 2: A Practical Approach Oxford University Press, Oxford, pp. 7, 19).
PCR assays that discriminate and identify alleles are important to genotype DNA samples for specific mutations (Livak (1999) Genetic Analysis: Biomolecular Engineering, 14:143-49; Mein etal (2000) Genome Research, 10:330-43). PCR assays also provide relative quantification of gene expression (Gibson etal (1996) Genome Research, 6:995-1001; Heid etal (1996) Genome Research, 6:986-94; Yang etal (1993) Anal. Biochem. 208:110-16; Germer etal (1999) Genome Research, 9:72-78).
Fluorescence-based approaches to provide end-point or real-time measurements of PCR amplification products (amplicons) (Holland etal, (1991) Proc. Natl. Acad. Sci., 88:7276-80) have either employed intercalating dyes, e.g. ethidium bromide, to indicate the amount of double stranded DNA present (Gelfand etal, U.S. Pat. No. 5,210,015) or probes containing reporter-quencher pairs (“TaqMan®”, 5′ nuclease assay) that are cleaved during amplification to release a fluorescent signal proportional to the amount of double stranded DNA present (Livak etal, U.S. Pat. No. 5,538,848; Gelfand etal, U.S. Pat. No. 5,804,375).
Parallel control tests that confirm conditions for amplification of the target are desirable. PCR is often plagued by false positives due to template contamination from adjacent wells, pipetting errors, or aerosol transmission, especially in high density or high-throughput formats, such as 96-, 384-well, or higher density microtitre plate, or other array configurations. In addition, PCR suffers from false negative results when enzyme inhibitors are present in the target samples or when reagents are missing or degraded. Control amplification reactions are desirable for (i) normalization of quantification results, (ii) detection of amplification inhibitors in the target and other reagents, and (iii) to establish background signal levels. Positive amplification control tests for PCR give a detectable amplicon derived from a control template that is separate and distinct from the target. Detection of the positive control amplicon indicates that amplification is viable and operative within the reaction chamber. Positive amplification control tests which give no detectable product from the control components, indicate conditions within the reaction chamber that do not allow amplification, such as contaminants that inhibit PCR.
Internal control PCR is conducted in the same vessel, concurrently with PCR of the target sample polynucleotide (Coen, D. “Quantification of DNAs by the Polymerase Chain Reaction Using an Internal Control” in The Polymerase Chain Reaction (1994) Mullis etal, Eds., Birkhauser, Boston, Mass., pp. 89-96; Coen, D. “Quantitation of rare DNAs by polymerase chain reaction” in Current Protocols in Molecular Biology (1990) Ausubel, etal, Eds. Greene Publ. Assoc. and Wiley-Interscience). Amplification of an internal control polynucleotide (ICP) with primers common to the target polynucleotide and the internal control polynucleotide, gives verification of true or false negatives, i.e. if target is not detected (Gibson etal (1996) Genome Research 6:995-1001). An ICP is often an endogenous region of the target polynucleotide sample, i.e. from the same source, genome, chromosome, gene, plasmid, or fragment as the target, thus normalizing for variation in the amount of target polynucleotide. An ICP may be an endogenous RNA or DNA sequence which is present in each experimental sample as isolated. An ICP may be an exogenous or foreign sequence of RNA or DNA which is spiked in to the target sample at a known concentration. The exogenous ICP may be an in vitro construct, used to distinguish true target negatives from PCR inhibition and normalize for differences in efficiency of sample extraction cDNA synthesis (Aoyagi, U.S. Pat. No. 5,952,202).
A pervasive difficulty with internal controls is keeping amplification of the control polynucleotide from interfering with target amplification or detection of the product (Reischl etal (1995) “Quantitative PCR” in Molecular Biotechnology, Vol. 3, Humana Press Inc., pp.55-71). Endogenous ICP are subject to amplification inhibitors and can therefore give a false negative signal. Endogenous ICP also may have priming sites for target primers and therefore give a false positive signal. Where endogenous ICP systems share one or more primers with the target, exhaustion of the shared primers may lead to inaccurate PCR quantification and limited dynamic range. In view of the limitations and deficiencies of conventional controls for the quantification and detection of nucleic acid amplification products, non-gel based, external control PCR methods that provide positive indications of amplification are desirable.